Genetic Determinants of Hydrogen Sulfide Biosynthesis in Fusobacterium nucleatum Are Required for Bacterial Fitness, Antibiotic Sensitivity, and Virulence.

The Gram-negative anaerobe Fusobacterium nucleatum is a major producer of hydrogen sulfide (HS), a volatile sulfur compound that causes halitosis. Here, we dissected the genetic determinants of HS production and its role in bacterial fitness and virulence in this important member of the oral microbiome. F. nucleatum possesses four enzymes, CysK1, CysK2, Hly, and MegL, that presumably metabolize l-cysteine to HS, and CysK1 was previously shown to account for most HS production , based on correlations of enzymatic activities with gene expression at mid-log phase. Our molecular studies showed that and were highly expressed at the late exponential growth phase, concomitant with high-level HS production, while the expression levels of the other genes remained substantially lower during all growth phases. Although the genetic deletion of without supplementation with a CysK1-catalyzed product, lanthionine, caused cell death, the conditional Δ mutant and a mutant lacking were highly proficient in HS production. In contrast, a mutant devoid of showed drastically reduced HS production, and a mutant showed only minor deficiencies. Intriguingly, the exposure of these mutants to various antibiotics revealed that only the mutant displayed altered susceptibility compared to the parental strain: partial sensitivity to nalidixic acid and resistance to kanamycin. Most significantly, the mutant was attenuated in virulence in a mouse model of preterm birth, with considerable defects in the spread to amniotic fluid and the colonization of the placenta and fetus. Evidently, the l-methionine γ-lyase MegL is a major HS-producing enzyme in fusobacterial cells that significantly contributes to fusobacterial virulence and antibiotic susceptibility. Fusobacterium nucleatum is a key commensal anaerobe of the human oral cavity that plays a significant role in oral biofilm development and contributes to additional pathologies at extraoral sites, such as promoting preterm birth and colorectal cancer. Although F. nucleatum is known as a major producer of hydrogen sulfide (HS), its genetic determinants and physiological functions are not well understood. By a combination of bacterial genetics, biochemical methods, and models of infection, here, we demonstrate that the l-methionine γ-lyase MegL not only is a major HS-producing enzyme of F. nucleatum but also significantly contributes to the antibiotic susceptibility and virulence of this organism.